Estrogen-regulated mRNA degradation has been observed in multiple breast and uterine cancer cell lines. As a model system to examine this destabilization, an estrogen-regulated polysomal mRNA endonuclease (PMR-1) has been purified to homogeneity from Xenopus laevis liver. Biochemical and immunological data described in this proposal provide convincing evidence that PMR-1 is phosphorylated. The primary hypothesis of this proposal is that phosphorylation is involved in the activation of PMR-1. Phosphatase treatment of hepatocyte extracts grown in the presence or absence of estrogen, and 2D gel electrophoresis will examine the kinetics of PMR-1 phosphorylation in response to estrogen. This data will be compared to the estrogen-activated degradation of albumin mRNA to provide evidence that phosphorylation activates PMR-1. Electrospray mass spectroscopy will be used to precisely map the location of any phosphoamino acids. Density grandient centrifugation of de-phosphorylated PMR-1/polysome mixtures will test whether phosphorylation modulates the affinity of PMR-1 with polysomes. A yeast expression system is described which will be used to analyze the functional effect of PM-1 phosphorylation. Site-directed mutants will be examined for their ability to associate with polysomes, and with specific proteins currently being identified by another member of the laboratory using the yeast "two- hybrid" assay system.